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[Preprint]. 2023 May 18:2023.05.18.541325.
doi: 10.1101/2023.05.18.541325.

Dynamic nuclear polarization illuminates key protein-lipid interactions in the native bacterial cell envelope

James E Kent  1 , Bryce E Ackermann  2 , Galia T Debelouchina  2 , Francesca M Marassi  1   3
Affiliations

Dynamic nuclear polarization illuminates key protein-lipid interactions in the native bacterial cell envelope

James E Kent et al. bioRxiv. .

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Abstract

Elucidating the structure and interactions of proteins in native environments has become a fundamental goal of structural biology. Nuclear magnetic resonance (NMR) spectroscopy is well suited for this task but often suffers from low sensitivity, especially in complex biological settings. Here, we use a sensitivity-enhancement technique called dynamic nuclear polarization (DNP) to overcome this challenge. We apply DNP to capture the membrane interactions of the outer membrane protein Ail, a key component of the host invasion pathway of Yersinia pestis . We show that the DNP-enhanced NMR spectra of Ail in native bacterial cell envelopes are well resolved and enriched in correlations that are invisible in conventional solid-state NMR experiments. Furthermore, we demonstrate the ability of DNP to capture elusive interactions between the protein and the surrounding lipopolysaccharide layer. Our results support a model where the extracellular loop arginine residues remodel the membrane environment, a process that is crucial for host invasion and pathogenesis.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1.
Figure 1.. DNP signal enhancement of the 15N and 13C MAS solid-state NMR spectra of Ail in E. coli cell envelopes.
(A-C) One dimensional spectra acquired with (A) 1H-15N CP, (B) 1H-13C CP, or (C) 1H-15N-13C double CP, and with (green) or without (black) microwave irradiation. Signal enhancement factors (εon/off) were measured as the ratio of signal intensity observed with and without microwave irradiation. (D, E) Two dimensional 13C/13C correlation spectrum of Ail in E. coli cell envelopes acquired with DNP (green). The spectra from Ail in E. coli cell envelopes (black) and Ail in liposomes (red), acquired without DNP are shown for comparison and were described previously (5). The yellow rectangle marks LPS-related correlations.
Figure 2.
Figure 2.. Two-dimensional NCA spectrum of 15N,13C-Ail in E. coli cell envelopes acquired with DNP (green).
Resolved assigned peaks are marked. Asterisks denote new unassigned peak observed with DNP. The spectrum from Ail in E. coli cell envelopes (black) acquired without DNP is shown for comparison and was described previously (5).
Figure 3.
Figure 3.. Two-dimensional NHHC spectra acquired with DNP.
(A-C) Spectra were acquired for (15N,13C)-Ail(+) (green) or (15N,13C)-Ail(−) (pink) E. coli cell envelopes. Spectral regions of correlations are marked (gold boxes). One-dimensional slices (B, C) were taken at specific 15N chemical shifts (dashed lines) of sidechain N from Arg and Lys. (D) Snapshot of Ail obtained after 1.5 μs of MD simulation in a Y. pestis outer membrane showing that Arg27 in the LPS-recognition motif establishes multiple interactions with lipid A of an LPS molecule. MD simulations were described previously (23).
See this image and copyright information in PMC

References

    1. Theillet F. X. (2022) In-Cell Structural Biology by NMR: The Benefits of the Atomic Scale, Chemical reviews 122, 9497–9570. - PubMed
    1. Luchinat E., Cremonini M., and Banci L. (2022) Radio Signals from Live Cells: The Coming of Age of In-Cell Solution NMR, Chemical reviews 122, 9267–9306. - PMC - PubMed
    1. Frederick K. K., Michaelis V. K., Corzilius B., Ong T. C., Jacavone A. C., Griffin R. G., and Lindquist S. (2015) Sensitivity-enhanced NMR reveals alterations in protein structure by cellular milieus, Cell 163, 620–628. - PMC - PubMed
    1. Kaplan M., Narasimhan S., de Heus C., Mance D., van Doorn S., Houben K., Popov-Celeketic D., Damman R., Katrukha E. A., Jain P., Geerts W. J. C., Heck A. J. R., Folkers G. E., Kapitein L. C., Lemeer S., van Bergen En Henegouwen P. M. P., and Baldus M. (2016) EGFR Dynamics Change during Activation in Native Membranes as Revealed by NMR, Cell 167, 1241–1251 e1211. - PubMed
    1. Kent J. E., Fujimoto L. M., Shin K., Singh C., Yao Y., Park S. H., Opella S. J., Plano G. V., and Marassi F. M. (2021) Correlating the Structure and Activity of Y. pestis Ail in a Bacterial Cell Envelope, Biophys. J. 120, 453–462. - PMC - PubMed

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